Lactoferrin and neuronal health and development in the infant gut

ABSTRACT

The present invention relates generally to the field of neuronal health, neuronal protection and neuronal development. One embodiment of the present invention relates to a composition that can be used for the treatment or prevention of a delayed development of the enteric nervous system. Neuronal cells in the gut can be protected. Disorders linked to a delayed development of the enteric nervous system and/or to an impaired enteric nervous system can be treated or prevented by the administration of lactoferhn containing compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage of International ApplicationNo. PCT/EP2010/056234, filed on May 7, 2010, which claims priority toEuropean Patent Application No. 09159968.8, filed on May 12, 2009, theentire contents of which are being incorporated herein by reference.

The present invention relates generally to the field of neuronal health,neuronal protection and neuronal development. One embodiment of thepresent invention relates to a composition that can be used for thetreatment or prevention of a delayed development of the enteric nervoussystem. Neuronal cells in the gut can be protected. Theirdevelopment/growth can be promoted, e.g., if a neuronal retardation hasto be recovered. Disorders linked to a delayed development of theenteric nervous system and/or to an impaired enteric nervous system canbe treated or prevented by the administration of lactoferrin containingcompositions according to the present invention.

The nervous system is a highly complex network composed of neuronal andglial cells. It is present in all mammalian species. The nervous systemis composed of the central nervous system (brain and spinal cord) andthe peripheral nervous system (somatic, autonomous and enteric nervoussystem). The central nervous system drives the cognitive functions(memory, attention, perception, action, etc).

The central nervous system drives the cognitive functions (memory,attention, perception, action, etc). Together with the peripheralnervous system, it has a fundamental role in the control of behaviour.The somatic nervous system is responsible for coordinating the body'smovements (under conscious control). The autonomous nervous systemmaintains homeostasis in the body activities without conscious control(heart rate, etc). Finally, and part of this latest, the enteric nervoussystem, which directly controls the gastrointestinal tract functionssuch as the intestinal barrier, the motility, the absorption, thedigestion and the secretions, contributing as such to the protection ofthe intestine from any type and to digestive comfort.

The nervous system develops during gestation and then refines to amature, functional network during the post natal period.

Immaturity or delayed maturation of the nervous system leads to delayedestablishment of the important biological functions that it regulates.In particular, it leads to intestinal dysfunctions after birth,including a low digestive/absorptive capacity, gastrointestinal reflux,a slower intestinal transit, a weak intestinal barrier function, leadingto food intolerance (therefore need of parenteral nutritional support),gut discomfort with harder stools, and increased infection and allergyrisks.

It was an object of the present invention to improve the state of theart and to provide a composition that is based on natural ingredientsand that allows supporting the development of the nervous system in thegut and to protect it.

This object was achieved by the subject matter of the independentclaims.

The present inventors were able to demonstrate that lactoferrin, forexample a composition supplemented with lactoferrin, can be used tosupport the development of neuronal cells and to protect them.

It could further be shown that the administration of lactoferrin allowsit to increase the neuron density and neuron survival.

Lactoferrin (LF), also known as lactotransferrin (LTF), is a globularmultifunctional protein that is known to exhibit an antimicrobialactivity and is a part of the innate defense, mainly at mucoses.

Lactoferrin may be found for example in milk and whey and in manymucosal secretions such as tears and saliva. As such, Lactoferrin may bepurified, e.g., from milk or may be produced recombinantly.

The present invention relates to lactoferrin obtainable from any source.

Lactoferrin from milk or whey, for example, has the advantage that it isa natural ingredient obtained from a food-grade composition and canconsequently be used as enriched fraction of the food compositionwithout further purification.

Recombinantly obtained lactoferrin has the advantage that it can beproduced easily in high concentrations.

Human colostrum has a relatively high concentration of lactoferrin,followed by human milk, then cow milk.

The present inventors have found that lactoferrin or lactoferrinenriched compositions may be used to protect neuronal cells againstdegeneration. Such degeneration may follow, for example, any stresssituations, such as those affecting the fetus (in utero) or the newborns(hypoxia-Ischemia at birth, oxygen therapy and hyperoxia, inflammation,need for parenteral support, etc). Lactoferrin was found to promoteneuronal survival and/or limit or prevent neuronal death of entericneuronal cells, and to promote neuronal growth which is important, e.g.,in developmental processes.

In infants the lactoferrin and/or the lactoferrin containingcompositions of the present inventions may be used to protect theenteric nervous system from any stress, e.g., occurring during theneuronal development period, and—consequently—to limit and/or preventstress-induced neuronal growth retardation and associated intestinaldysfunctions.

For the purpose of the present invention, the term “infant” includeschildren and comprises subjects in the age range from 0-14 years.

A human infant less than a month old is a newborn or a neonate. The term“newborn” includes premature infants, postmature infants and full termnewborns. Upon reaching the age of one or beginning to walk, infants arereferred to as “toddlers” (generally 12-36 months).

Lactoferrin and/or the composition of the present invention may beadministered, for example, to

-   -   infants    -   neonates    -   mothers during pregnancy and/or lactation    -   premature or term-born infants having experienced an        intrauterine growth retardation that may occur following any        adverse events during the gestation (e.g., smoking of the        mother, medication of the mother, low placenta quality, abnormal        placenta positioning, malnutrition of the mother and the fetus,        etc)    -   premature infants without any intrauterine growth retardation    -   low birth weight/very low birth weight infants    -   non organic failure to thrive infants    -   any neonate and infant showing nervous system growth retardation        following for example hypoxemia-ischemia at birth or any other        adverse event, and/or    -   any neonate and infant showing gastrointestinal dysfunctions        (digestive disorders, motility disorders, gastrointestinal        reflux, slow gastrointestinal transit, oral feeding        intolerance), Hirschsprung's disease, and inflammation affecting        the gastrointestinal tract (such as Necrotisis enterocolitis),        obstruction pathologies

Lactoferrin or the composition of the present invention can therefore beadministered to the infant and/or to the mother during the gestationand/or lactation period.

Consequently, one embodiment of the present invention is an ingestiblecomposition enriched in lactoferrin.

Enriched means that lactoferrin was either added to the composition, sothat the resulting lactoferrin content of the composition is higher thanthe lactoferrin content of the composition without lactoferrin addition,or that the composition was treated in a way to concentrate the naturallactoferrin content in a composition.

Lactoferrin may also be provided as pure compound.

Alternatively, lactoferrin may be provided as a lactoferrin enrichedfraction, for example a lactoferrin enriched milk or whey fraction.

As milk or whey source bovine milk, human milk, goat milk, camel milk,horse milk and/or donkey milk may be used, for example. Colostrum may beused as well.

In therapeutic applications, compositions are administered in an amountsufficient to at least partially cure or arrest the symptoms of thedisorder and/or its complications.

An amount adequate to accomplish this is defined as “a therapeuticallyeffective dose”. Amounts effective for this purpose will depend on anumber of factors known to those of skill in the art such as theseverity of the disorder and the weight and general state of thepatient. In prophylactic applications, compositions according to theinvention are administered to a patient susceptible to or otherwise atrisk of a particular disorder in an amount that is sufficient to atleast partially reduce the risk of developing a disorder. Such an amountis defined to be “a prophylactic effective dose”. Again, the preciseamounts depend on a number of patient specific factors such as thepatient's state of health and weight.

Lactoferrin may be administered in the framework of the presentinvention in a therapeutically effective dose and/or in a prophylacticeffective dose.

Typical lactoferrin enriched compositions may comprise lactoferrin in anamount of at least 1.6 g/L.

For example, the composition of the present invention may containlactoferrin in a concentration of at least 0.75% (w/w), preferably atleast 1% (w/w).

In one embodiment, the composition is to be administered in an amountcorresponding to an ingestion of at least 0.25 g lactoferrin, preferablyat least 0.5 g lactoferrin more preferably at least 1 g lactoferrin perday per kg body weight.

For example, the composition may be consumed in an amount correspondingto at least 1 g lactoferrin/kg body weight/day intake for pregnantand/or lactating mothers.

The composition may also be consumed in an amount corresponding to atleast 200 mg lactoferrin/kg body weight/day intake for the children.

Lactoferrin may be present in the composition in a concentration of atleast 0.01 g per 100 kcal, preferably of at least 0.1 g per 100 kcal.For example, lactoferrin may be present in the composition in the rangeof about 0.01 g-100 g, preferably 0.1 g-50 g, even more preferred 2 g-25g per 100 kcal of the composition.

Lactoferrin may also be used in combination with other compounds, suchas sialic acid and/or iron, for example.

Sialic acid is a generic term for the N- or O-substituted derivatives ofneuraminic acid, a monosaccharide with a nine-carbon backbone.

Any sialic acid may be used for the purposes of the present invention.However, it is preferred if the sialic acid has the following formula

-   R1=H, acetyl, lactyl, methyl, sulfate, phosphate, anhydro, sialic    acid, fucose, glucose, or galactose-   R2=N-acetyl, N-glycolyl, amino, hydroxyl, N-glycolyl-O-acetyl, or    N-glycolyl-O-methyl-   R3=H, galactose, N-acetylglucosamine, N-acetylgalactosamine, sialic    acid, or N-glycolylneuraminic acid

R1 may be selected from the group consisting of H, acetyl, lactyl,methyl, sulfate, phosphate, anhydrosialic acid, fucose, glucose and/orgalactose.

R2 may be selected from the group consisting of N-acetyl, N-glycolyl,amino, hydroxyl, N-glycolyl-O-acetyl, and/or N-glycolyl-O-methyl.

R3 may be selected from the group consisting of H, galactose,N-acetylglucosime, N-acetylgalactosamine, sialic acid, and/orn-glycolylneuraminic acid.

The groups in position R1 may be identical or may differ from eachother.

For example, the sialic acid may be N-acetylneuraminic acid with R1=H,R2=N-acetyl and R3=H. According to a further embodiment of the presentinvention the sialic acid may selected from the group consisting of2-keto-5-acetamido-3,5-dideoxy-d-glycero-d-galactononulosonic acid(Neu5Ac) and 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN) ormixtures thereof.

Sialic acid as used in the present invention comprisesN-Acetylneuraminic acid, which has the following synonyms andabbreviations: o-Sialic acid;5-Acetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid;5-Acetamido-3,5-dideoxy-D-glycero-D-galactonulosonic acid; Aceneuramicacid; N-acetyl-neuraminate; N-Acetylneuraminic acid; NANA, and Neu5Ac.

A particular preferred lactoferrin containing composition may containsialic acid in an amount in the range of 100 mg/100 g (w/w) to 1000mg/100 g (w/w) of the composition, for example in the range of 500mg/100 g (w/w) to 650 mg/100 g (w/w) of the composition.

The composition of the present invention may for example comprise atleast about 0.001 weight-% sialic acid. In further embodiments of thepresent invention, the composition may comprise at least about 0.005weight-%, or at least about 0.01 weight-% of sialic acid.

Alternatively or additionally the lactoferrin containing composition maycontain iron in an amount in the range of about 1 mg/100 g (w/w) to 100mg/100 g (w/w) of the composition, for example 10 mg/100 g (w/w) to 30mg/100 g (w/w) of the composition.

One lactoferrin containing composition may contain for example about 852mg/100 g (w/w) sialic acid and 22 mg/100 g (w/w) iron.

The lactoferrin containing composition of the present invention may havea caloric density in the range of 30 kcal/100 g-1000 kcal/100 g of thecomposition, preferably 50 kcal/100 g-450 kcal/100 g of the composition.It may for example have a caloric density of about 400 kcal/100 g.

The nature of the composition is not particularly limited. It ispreferably a composition for oral or enteral administration.

The composition may be for example selected from the group consisting offood products, animal food products, pharmaceutical compositions,nutritional formulations, nutraceuticals, drinks, food additives, andinfant feeding formulas.

In one typical embodiment of the present invention, the composition willcontain a protein source, a lipid source and a carbohydrate source.

The composition comprises a protein source which may be present in therange of between 1.4 and 100 g/100 kcal, preferably between 2 and 6.0g/100 kcal of the composition. Since lactoferrin is a protein it shouldbe considered a part of the protein source.

The type of protein is not believed to be critical to the presentinvention. Thus, protein sources based on whey, casein and mixturesthereof may be used, for example. As far as whey proteins are concerned,acid whey or sweet whey or mixtures thereof may be used as well asalpha-lactalbumin and beta-lactoglobulin in whatever proportions aredesired. The whey protein may be modified sweet whey. Sweet whey is areadily available by-product of cheese making and is frequently used inthe manufacture of infant formulas based on cows' milk. However, sweetwhey includes a component which is undesirably rich in threonine andpoor in tryptophan called caseino-glyco-macropeptide (CGMP). Removal ofthe CGMP from sweet whey results in a protein with a threonine contentcloser to that of human milk. This modified sweet whey may then besupplemented with those amino acids in respect of which it has a lowcontent (principally histidine and tryptophan). A process for removingCGMP from sweet whey is described in EP 880902 and an infant formulabased on this modified sweet whey is described in WO 01/11990. Theproteins may be intact or hydrolysed or a mixture of intact andhydrolysed proteins. It may be desirable to supply partially hydrolysedproteins (degree of hydrolysis between 2 and 20%), for example forsubjects believed to be at risk of developing cows' milk allergy. Ifhydrolysed proteins are required, the hydrolysis process may be carriedout as desired and as is known in the art. For example, a whey proteinhydrolysate may be prepared by enzymatically hydrolysing the wheyfraction in two steps as described in EP 322589. For an extensivelyhydrolysed protein, the whey proteins may be subjected to triplehydrolysis using Alcalase 2.4L (EC 940459), then Neutrase 0.5L(obtainable from Novo Nordisk Ferment AG) and then pancreatin at 55° C.If the whey fraction used as the starting material is substantiallylactose free, it is found that the protein suffers much less lysineblockage during the hydrolysis process. This enables the extent oflysine blockage to be reduced from about 15% by weight of total lysineto less than about 10% by weight of lysine; for example about 7% byweight of lysine which greatly improves the nutritional quality of theprotein source.

Whey protein is known to provide several health benefits. For example,it is easily digestible. The protein fraction in whey (approximately 10%of the total dry solids within whey) comprises several proteinfractions, for example beta-lactoglobulin, alpha-lactalbumin, bovineserum albumin and immunoglobulins. In one embodiment at least 50%,preferably at least 75%, even more preferred at least 85% by weight ofthe protein source is whey protein.

The compositions of the present invention may contain a lipid source.The lipid source may be any lipid or. Preferred fat sources include milkfat, palm olein, high oleic sunflower oil and high oleic safflower oil.The essential fatty acids linoleic and α-linolenic acid may also beadded as may small amounts of oils containing high quantities ofpreformed arachidonic acid and docosahexaenoic acid such as fish oils ormicrobial oils. The lipid source preferably has a ratio of n-6 to n-3fatty acids of about 5:1 to about 15:1; for example about 8:1 to about10:1.

If present, the lipid source may contribute to between 30 to 55% of thetotal energy of the composition.

The compositions of the present invention may contain a carbohydratesource. Any carbohydrate source may be used, such as lactose,saccharose, maltodextrin, starch and mixtures thereof.

A carbohydrate source may contribute to between 35 and 65% of the totalenergy of the composition.

For example a composition of the present invention may comprise proteinin the range of about 1.8 to 3.0 g/100 kcal, lipids in the range ofabout 4.4 to 6.5 g/100 kcal and/or carbohydrates in the range of about1.7 to 12 g/100 kcal.

If the composition is liquid, its energy density may be between 60 and75 kcal/100 ml.

If the composition is solid, its energy density may be between 60 and 75kcal/100 g.

The compositions of the present invention may also contain all vitaminsand minerals understood to be essential in the daily diet and innutritionally significant amounts. Minimum requirements have beenestablished for certain vitamins and minerals. Examples of minerals,vitamins and other nutrients optionally present in the infant formulainclude vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12,vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol,niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine,iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium,selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals areusually added in salt form. The presence and amounts of specificminerals and other vitamins will vary depending on the numerous factors,such as age weight and condition of the person or animal the compositionis administered to.

The compositions may also comprise at least one probiotic bacterialstrain. A probiotic is a microbial cell preparation or components ofmicrobial cells with a beneficial effect on the health or well-being ofthe host. Suitable probiotic bacterial strains include Lactobacillusrhamnosus ATCC 53103 obtainable from Valio Oy of Finland under the trademark LGG, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracaseiCNCM 1-2116, Lactobacillus reuteri ATCC 55730 and Lactobacillus reuteriDSM 17938 obtainable from BioGaia AB, Bifidobacterium lactis CNCM 1-3446sold inter alia by the Christian Hansen company of Denmark under thetrade mark Bb12 and Bifidobacterium longum ATCC BAA-999 sold by MorinagaMilk Industry Co. Ltd. of Japan under the trade mark BB536. The amountof probiotic, if present, likewise preferably varies as a function ofthe age of the person or animal. Generally speaking, the probioticcontent may increase with increasing age of the infant for example from10³ to 10¹² cfu/g formula, more preferably between 10⁴ and 10⁸ cfu/gformula (dry weight).

The compositions may also contain at least one prebiotic in an amount of0.3 to 10%. A prebiotic is a non-digestible food ingredient thatbeneficially affects the host by selectively stimulating the growthand/or activity of one or a limited number of bacteria in the colon, andthus improves host health. Such ingredients are non-digestible in thesense that they are not broken down and absorbed in the stomach or smallintestine and thus pass intact to the colon where they are selectivelyfermented by the beneficial bacteria. Examples of prebiotics includecertain oligosaccharides, such as fructooligosaccharides (FOS) andgalactooligosaccharides (GOS). A combination of prebiotics may be usedsuch as 90% GOS with 10% short chain fructo-oligosaccharides such as theproduct sold under the trade mark Raftilose® or 10% inulin such as theproduct sold under the trade mark Raftiline®.

A particularly preferred prebiotic is a mixture ofgalacto-oligosaccharide(s), N-acetylated oligosaccharide(s) andsialylated oligosaccharide(s) in which the N-acetylatedoligosaccharide(s) comprise 0.5 to 4.0% of the oligosaccharide mixture,the galacto-oligosaccharide(s) comprise 92.0 to 98.5% of theoligosaccharide mixture and the sialylated oligosaccharide(s) comprise1.0 to 4.0% of the oligosaccharide mixture. This mixture is hereinafterreferred to as “CMOS-GOS”. Preferably, a composition for use accordingto the invention contains from 2.5 to 15.0 wt % CMOS-GOS on a dry matterbasis with the proviso that the composition comprises at least 0.02 wt %of an N-acetylated oligosaccharide, at least 2.0 wt % of agalacto-oligosaccharide and at least 0.04 wt % of a sialylatedoligosaccharide.

Suitable N-acetylated oligosaccharides include GalNAcα1,3Galβ1,4Glc andGalβ1,6GalNAcα1,3Galβ1,4Glc. The N-acetylated oligosaccharides may beprepared by the action of glucosaminidase and/or galactosaminidase onN-acetyl-glucose and/or N-acetyl galactose. Equally, N-acetyl-galactosyltransferases and/or N-acetyl-glycosyl transferases may be used for thispurpose. The N-acetylated oligosaccharides may also be produced byfermentation technology using respective enzymes (recombinant ornatural) and/or microbial fermentation. In the latter case the microbesmay either express their natural enzymes and substrates or may beengineered to produce respective substrates and enzymes. Singlemicrobial cultures or mixed cultures may be used. N-acetylatedoligosaccharide formation can be initiated by acceptor substratesstarting from any degree of polymerisation (DP) from DP=1 onwards.Another option is the chemical conversion of keto-hexoses (e.g.fructose) either free or bound to an oligosaccharide (e.g. lactulose)into N-acetylhexosamine or an N-acetylhexosamine containingoligosaccharide as described in Wrodnigg, T. M.; Stutz, A. E. (1999)Angew. Chem. Int. Ed. 38:827-828. Suitable galacto-oligosaccharidesinclude Galβ1,6Gal, Galβ1,6Galβ1,4Glc Galβ1,6Galβ1,6Glc,Galβ1,3Galβ1,3Glc, Galβ1,3Galβ1,4Glc, Galβ1,6Galβ1,6Galβ1,4Glc,Galβ1,6Galβ1,3Galβ1,4Glc Galβ1,3Galβ1,6Galβ1,4Glc,Galβ1,3Galβ1,3Galβ1,4Glc, Galβ1,4Galβ1,4Glc andGalβ1,4Galβ1,4Galβ1,4Glc. Synthesised galacto-oligosaccharides such asGalβ1,6Galβ1,4Glc Galβ1,6Galβ1,6Glc, Galβ1,3Galβ1,4Glc,Galβ1,6Galβ1,6Galβ1,4Glc, Galβ1,6Galβ1,3Galβ1,4Glc andGalβ1,3Galβ1,6Galβ1,4Glc, Galβ1,4Galβ1,4Glc and Galβ1,4Galβ1,4Galβ1,4Glcand mixtures thereof are commercially available under the trade marksVivinal® and Elix'or®. Other suppliers of oligosaccharides are DextraLaboratories, Sigma-Aldrich Chemie GmbH and Kyowa Hakko Kogyo Co., Ltd.Alternatively, specific glycoslytransferases, such asgalactosyltransferases may be used to produce neutral oligosaccharides.

Suitable sialylated oligosaccharides include NeuAcα2,3Galβ1,4Glc andNeuAcα2,6Galβ1,4Glc. These sialylated oligosaccharides may be isolatedby chromatographic or filtration technology from a natural source suchas animal milks. Alternatively, they may also be produced bybiotechnology using specific sialyltransferases either by enzyme basedfermentation technology (recombinant or natural enzymes) or by microbialfermentation technology. In the latter case microbes may either expresstheir natural enzymes and substrates or may be engineered to producerespective substrates and enzymes. Single microbial cultures or mixedcultures may be used. Sialyl-oligosaccharide formation can be initiatedby acceptor substrates starting from any degree of polymerisation (DP)from DP=1 onwards.

The compositions may optionally contain other substances which may havea beneficial effect, such as nucleotides, nucleosides, and the like.

The compositions, for example an infant formula, for use in theinvention may be prepared in any suitable manner. For example, an infantformula may be prepared by blending together the protein source, thecarbohydrate source, and the fat source in appropriate proportions. Ifused, the emulsifiers may be included in the blend. The vitamins andminerals may be added at this point but are usually added later to avoidthermal degradation. Any lipophilic vitamins, emulsifiers and the likemay be dissolved into the fat source prior to blending. Water,preferably water which has been subjected to reverse osmosis, may thenbe mixed in to form a liquid mixture. The liquid mixture may then bethermally treated to reduce bacterial loads. For example, the liquidmixture may be rapidly heated to a temperature in the range of about 80°C. to about 110° C. for about 5 seconds to about 5 minutes. This may becarried out by steam injection or by heat exchanger; for example a plateheat exchanger. The liquid mixture may then be cooled to about 60° C. toabout 85° C.; for example by flash cooling. The liquid mixture may thenbe homogenised; for example in two stages at about 7 MPa to about 40 MPain the first stage and about 2 MPa to about 14 MPa in the second stage.The homogenised mixture may then be further cooled to add any heatsensitive components; such as vitamins and minerals. The pH and solidscontent of the homogenised mixture is conveniently standardised at thispoint. The homogenised mixture is transferred to a suitable dryingapparatus such as a spray drier or freeze drier and converted to powder.The powder should have a moisture content of less than about 5% byweight. If it is desired to add probiotic(s), they may be culturedaccording to any suitable method and prepared for addition to the infantformula by freeze-drying or spray-drying for example. Alternatively,bacterial preparations can be bought from specialist suppliers such asChristian Hansen and Morinaga already prepared in a suitable form foraddition to food products such as infant formula. Such bacterialpreparations may be added to the powdered infant formula by dry mixing.

Lactoferrin may be added at any stage during this procedure, but ispreferably added after the heating step.

The present invention extends to the use of lactoferrin for thepreparation of a composition for the treatment or prevention of adelayed development of the enteric nervous system, and/or to protect theenteric nervous system; as well as for the treatment and/or preventionof gastrointestinal disorders linked to a delayed development of theenteric nervous system, and/or an impaired enteric nervous system.

Lactoferrin or the composition of the present invention may also be usedto repair an impaired enteric nervous system.

Disorders linked to a delayed development of the enteric nervous system,and/or an impaired enteric nervous system include for example a lowdigestive and/or absorptive capacity, gastrointestinal reflux, a slowintestinal transit, a weak intestinal barrier function, enteral feedingintolerance, gut discomfort, hard stools.

In another embodiment of the present invention, the lactoferrincontaining composition may be used to treat or prevent a delayed neuralmigration.

The composition of the present invention can be used to increase theneuronal density and or the neuronal survival.

For the uses of the present invention it is essential that thecomposition contains lactoferrin or a compound that yields lactoferrinafter consumption. The composition does not have to be enriched inlactoferrin, although this may be preferable, since this way morelactoferrin can be administered in smaller volumes.

The lactoferrin may be used to prepare any kind of composition. It ispreferred, however, that the lactoferrin is provided as a composition inaccordance with what is described above.

To achieve this, the composition may be administered to mothers duringpregnancy, mothers during lactation, to premature or term born babies,IUGR infants, infants, toddlers, children and/or teenagers.

Those skilled in the art will understand that they can freely combineall features of the present invention described herein, withoutdeparting from the scope of the invention as disclosed. In particular,features described for the uses of the present invention may be appliedto composition of the present invention and/or to lactoferrin and viceversa.

Further advantages and features of the present invention are apparentfrom the following Examples and Figures.

FIG. 1 shows the percentage of positive NS20Y cells for neuriteoutgrowth in basal condition (untreated cells) and after treatment ofthe cells with either the neurotrophic factor CNTF (100 ng/mL, positivecontrol) or the lactoferrin enriched bovine milk fraction at differentconcentrations. Data are means±SEM, n=3 to 7 according to the group(Basal, n=7; CNTF, n=3; 1 ug/L, n=3; 10 ug/L, n=7; 100 ug/L, n=3; 1mg/L, n=3; 10 mg/L, n=5; 100 mg/L, n=7; 1 g/L, n=6). Data were comparedto the basal untreated group with the student t test. A difference wasconsidered significant when P<0.05.

FIG. 2 shows the release of neuron-specific enolase (NSE), a marker forneuronal cell death, by a primary culture of enteric neurons, followingH₂O₂ challenge and prevention with bovine milk Lactoferrin. Data aremean±SEM, n=8. A difference was considered significant when P<0.05

FIG. 3 shows the percentage of 7-AAD positive cells in cultured SHSY5Ycells, following H₂O₂ challenge in presence or not of differentconcentrations of bovine milk Lactoferrin ranging from 0.001 to 1 g/L.

FIG. 4 shows the nuclei morphology in the CA2-CA3 field of thehippocampus after DAPI staining.

EXAMPLES

Biological activity of lactoferrin enriched bovine milk fraction has aneffect on promoting neuronal cell survival and neurite outgrowth invitro.

The neurite outgrowth process comprises the outgrowth of axons fromneurons and is part of neuronal development. The impact of a fraction ofbovine milk enriched in lactoferrin on neurite outgrowth was measuredusing a well established and commonly used in vitro bioassay.

Briefly, NS20Y murine neuroblastoma cells (DSMZ) were thawed fromcryogenic storage, plated at a density of approximately 27×10³ cells percm² in tissue culture-treated flasks (Falcon) and expanded in thepresence of DMEM (Gibco) containing 10% FCS (Gibco) and 2 mM L-glutamine(Gibco). Two days after plating, the cells were detached from the flaskby mechanical agitation (tapping of the flask), and a single cellsuspension was obtained by passing the suspension several times througha flame-polished glass pipette. Cells were then plated onto 13 mm roundglass coverslips in the presence of DMEM containing 10% FCS and 2 mML-glutamine at a density of 2,000 cells per coverslip. The following daythe medium was switched to DMEM containing 0.5% FCS, 2 mM L-glutamine,and different concentrations of the milk fractions to be tested. One daylater cells were fixed with 4% paraformaldehyde and the coverslipsmounted on slides.

All coverslips were imaged with an Axioplan 2 microscope (Zeiss).Digital images were taken from 25 defined fields across the diameter ofthe coverslip (20× objective, Axiocam MRc, Zeiss). Cells were countedsystematically from the first field at the edge of the coverslip acrossthe coverslip until 100 cells had been counted. Cells were scored foreither positive or negative for neurite outgrowth. Positive cells forneurite outgrowth were considered if the axon-resembling projectionsemanating from the cell body reached a length greater than the cellbody.

A student t test was used to compare differences in the mean between onecontrol reference population and means from all other treatments in eachgroup.

The following concentrations of the lactoferrin enriched bovine milkfraction were tested: 1 μg/L, 10 μg/L, 100 μg/L, 1 mg/L, 10 mg/L, 100mg/L, and 1 g/L. A positive control (CNTF, ciliary neurotrophic factor,100 ng/mL), which is a well known neurotrophic factor previouslyreported to promote neurite outgrowth of different neuronal populations(Oyesiku and Wigston, 1996 (Oyesiku N M, Wigston D J: Ciliaryneurotrophic factor stimulates neunte outgrowth from spinal cordneurons. J Comp Neurol 1996; 364: 68-77). was performed. A basal controlconsisted of untreated cells. Results are shown in FIG. 1.

Protection of Neuronal Cells Against Stress

Rat primary cultures of enteric neuronal cells were seeded into wellsand incubated with different concentrations of bovinelactoferrin-enriched fraction for 48 h. After washing three times withphosphate buffer saline (sterile PBS, 37° C.), the cells were incubatedfor 12 hours in cell medium without lactoferrin and containing H₂O₂ orits vehicle (control). The protective effect of lactoferrin uponH₂O₂-induced neuronal cell death was evaluated by measuring the releaseof neuron-specific enolase (NSE) in the cell medium. After oxidativestress, the medium of the different groups were collected andcentrifuged for 10 min at 12,000 rpm (4° C.). The supernatant wascollected and the NSE released in the culture medium was quantified byimmunoradiometric assay. Results are expressed in ng/mL. As shown onFIG. 2, H₂O₂ induced a significant increase of NSE in the medium(p<0.05, n=8). Treatment of primary neuronal enteric cells withlactoferrin-enriched fraction significantly prevented the H₂O₂-inducedrelease of NSE (p<0.05, n=8) as shown on FIG. 2.

The neuroprotective property of bovine lactoferrin was confirmed using ahuman neuronal-like cell line (SH-SY5Y-neuroblastoma cells). Briefly,SH-SY5Y cells were plated for 24 h, and bovine lactoferrin-enrichedfraction was added to the culture media of cells at differentconcentrations for the following 48 h. H₂O₂ for 6 h or with its vehicle(control) directly added to the cell media. Cells were finally washedwith 0.1 M PBS before being harvested with trypsine-EDTA. Cellsuspension was then pooled with the supernatant and centrifuged for 5min at 2,000 rpm. After centrifugation, the pellet was resuspended in500 microliter of PBS 0.1M. Membrane permeability was evaluated by flowcytometry using the 7-AAD as fluorescent marker. For this, 200microliter of cell suspension were incubated with 7-AAD for 10 minbefore acquisition using BD FACS Array™ bioanalyser. This flowcytometric assay using 7-aminoactinomycin D (7-AAD) allowed todistinguish live (7-AAD negative) and late apoptotic/necrotic (7-AADpositive) SH-SY5Y cells in response to oxidative stress. Results aspresented in FIG. 3 were expressed as percentage of 7-AAD positive cellsper total number of cells. As shown in FIG. 3, H₂O₂ induced asignificant increase in the percentage of 7-AAD positive cells (p<0.05,n=6). Treatment of SH-SY5Y cells with lactoferrin prevented theH₂O₂-induced increase in percentage of 7-ADD positive cells, as shown onFIG. 3.

Lactoferrin improves neuron density and neuron survival. It protectsneuronal cells and delays neuronal cell death.

A morphological examination was conducted following MR acquisition.Contiguous sections at the level of the striatum, dorsal and lateralhippocampus were collected to assess cortical and hippocampalarchitecture and white matter injury. Specific cells types were labeledusing immunohistochemistry, in order to determine specific cellularresponses. Specific labeling of neurons (NeuN), astrocytes (GFAP) andradial glia (Nestin), in conjunction with markers of white mattermyelination (MBP), was performed. The brief methodology was thefollowings:

At P7 and P21, respectively, pups from each group were deeplyanesthetized using ketalar (50 mg/m1; 0.2-0.5 ml, i.p.). Animals wereperfused intracardially with 0.9% NaCl, then 4% paraformaldehyde. Brainswere removed, weighed and postfixed in 4% paraformaldehyde overnight,then 30% sucrose for 24 h minimum, and stored at −80° C. untilsectioned. Coronal sections (10 μm) at the level of the dorsalhippocampus were cut on a cryostat (Microm Cryo-Star HM 560M, MicromInternational, Germany). Three sections at 200 μm intervals werecollected from each animal.

Immunohistochemitry: Brain tissue was processed for immunoreactivity toMBP (1:400 brand city country) using the avidin-biotin peroxidasecomplex (ABC; Vector

Laboratories, Burlingame, Calif., USA). Sections were blocked in 4%bovine serum albumin (BSA brand city country), then incubated with theprimary antibody for 24 h at 4° C., after which they were incubated withthe secondary antibody (1:200 brand city country), then with theavidin-biotin complex (1:200, Vector Laboratories, Burlingame, Calif.,USA). Sections were reacted with the chromagen, 3,3-diaminobenzidine(DAB brand city country) in 0.01% hydrogen peroxide, then coverslipped.

The same protocol was used for fluorescence immunohistochemistry fornestin (1:500 brand city country), GFAP (1:400 brand city country), andNeuN (1:200 brand city country), except that sections were not incubatedin the avidin-biotin complex and DAB.

Each experimental group and their respective controls were stainedsimultaneously. When the primary antibody treatment was omitted,staining failed to occur.

Quantitative analyses were performed using MetaMorph® Imaging System(Meta Imaging Software, Molecular Devices Corporation, Pennsylvania,U.S.A). Values for each animal were pooled and a mean of means±SEM wascalculated for each group. Measurements were made on coded slidesblinded to the observer with the codes not being disclosed until theconclusion of analyses.

The results were the following and are shown in FIG. 4.

The histological analysis revealed that LF supplementation Dex pup (n=2)has significant increased the Nuclei morphology and neuron density inthe CA2-CA3 field of the hippocampus compared to the Dex control pup atP7. A decrease in neuronal density in the cortex at P7 suggests neuronalloss. The neuronal density is similar to the normal control vehiclegroup (FIG. 4). Lactoferrin given in this particular developmental timeframe will influence neuronal density in the hippocampus and area ofgreat vulnerability for undernutrition and stress related abnormalities.This implies that LF administration increases neuron survival and neuronprotection, for example in a young IUGR rat.

Lactoferrin supplementation decreased the colonic gene expression ofSOX10 and CRHbp at postnatal day 21:

Colonic tissues of rat pups treated with dexamethasone and havingreceived or not an oral supplementation of either Lactoferrin or of acontrol isonitrogenous amino acid mixture during lactation tillpostnatal day 21 were collected. Total RNAs were extracted using theTriPure method. 0.5 ug RNA were reverse-transcribed using oligo-dT andthe SuperScript kit and a realtime PCR (Applied Biosystem, TaqMan PCR7900 HT) was performed. The genes of interest were normalized with thehousekeeping gene expression of 18S. After normalization, the percentageof change between the 2 groups were calculated (expressed in percent.n=6). Results showed that the gene expression of SOX10 was reduced by30% in the group of pups having received Lactoferrin as compared to thecontrol group. SOX10 is a marker for glial and neuronal progenitor cells(Wegner M, Stolt CC. From stem cells to neurons and glia: a Soxist'sview of neural development. Trends Neurosci 2005 25(11):583-8; Young etal., Acquisition of neuronal and glial markers by neural crest-derivedcells in the mouse intestine, The Journal of Comparative Neurology 2003456:1-11.). It is highly present in embryonic stages and early postnataldevelopment phases and its expression then decreases during thematuration process. Our results suggest an improved maturation ofneuronal progenitor cells to mature/differentiated neuronal cells of theenteric nervous system in the lactoferrin group at postnatal day 21. Ourresults also show a 60% reduction of the colonic gene expression of thecorticotropin releasing hormone binding protein (CRHbp) in thelactoferrin group as compared to controls. CRHbp, by binding tocorticotropin releasing hormone (CRH), limits the availability of freeand active CRH, and therefore reduce its stimulatory effect on colonicmotility (Saesholtz et al., Mouse models of altered CRH-binding proteinexpression. Peptides 2001. 22(5):743-51). The 60% reduction in geneexpression of CRHbp observed in our study may contribute to increase thefree and active colonic CRH availability which may promote colonicmotility function in these immature pups.

The invention claimed is:
 1. Ingestible composition comprisinglactoferrin in a concentration of 0.1 g-100 g/100 kcal of thecomposition, the composition comprising a source of protein, a source oflipids, and a source of carbohydrates and having a form of an infantfeeding formula, the source of protein present in an amount between 1.4and 4.0 g/100 kcal of the composition, the composition comprising atleast about 70 mg/L of total sialic acid, at least about 0.1% omega-3fatty acid by weight of total fatty acids, and at least about 0.25%omega-6 fatty acids by weight of the total fatty acids, and the sialicacid is selected from the group consisting of2-keto-5-acetamido-3,5-dideoxy-d-glycero-d-galactononulosonic acid(Neu5Ac), 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN) andcombinations thereof.
 2. Composition according to claim 1, wherein thelactoferrin is provided as a milk or whey fraction enriched inlactoferrin.
 3. Composition according to claim 1, wherein over 50% byweight of the protein source is whey protein.
 4. Composition accordingto claim 1, wherein the lipid source contributes between 30 to 55% ofthe total energy of the composition.
 5. Composition according to claim1, wherein the lactoferrin is present in a concentration in the range ofabout 2 g-25 g per 100 kcal.
 6. Composition according to claim 1,wherein the composition is to be administered in an amount correspondingto an ingestion of at least 0.01 g lactoferrin per kg body weight perday.
 7. A method for the promotion of the development of the entericnervous system comprising administering a composition comprisingLactoferrin according to claim 1 to an individual in need of same.
 8. Amethod for the repair of an impaired enteric nervous system comprisingadministering a composition comprising Lactoferrin according to claim 1to an individual in need of same.
 9. A method for treating disorderslinked to a delayed development of the enteric nervous system, and/or animpaired enteric nervous system comprising administering a compositioncomprising Lactoferrin according to claim 1 to an individual in need ofsame.
 10. Method in accordance with claim 9, wherein the disorder isselected form the group consisting of a low digestive and/or absorptivecapacity, gastrointestinal reflux, a slow intestinal transit, a weakintestinal barrier function, food intolerance, gut discomfort, and hardstools.
 11. Method of claim 7, wherein the individual is selected fromthe group consisting of mothers during pregnancy, mothers duringlactation, premature or term born babies, infants, toddlers, childrenand teenagers.
 12. Method of claim 8, wherein the individual is selectedfrom the group consisting of mothers during pregnancy, mothers duringlactation, premature or term born babies, infants, toddlers, childrenand teenagers.
 13. Method of claim 9, wherein the individual is selectedfrom the group consisting of mothers during pregnancy, mothers duringlactation, premature or term born babies, infants, toddlers, childrenand teenagers.
 14. Composition according to claim 1, wherein thecarbohydrate source contributes between 35 and 65% of the total energyof the composition.
 15. Method of claim 7, wherein the lactoferrin ispresent in the composition in a concentration of 2 g-25 g/100 kcal ofthe composition.
 16. Method of claim 7, wherein the composition isadministered to provide at least 0.2 g lactoferrin per kg of body weightof the individual per day.
 17. Method of claim 7, wherein thecomposition is administered to provide at least 1 g lactoferrin per kgof body weight of the individual per day.
 18. Method of claim 8, whereinthe lactoferrin is present in the composition in a concentration of 2g-25 g/100 kcal of the composition.